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1.
Adv Exp Med Biol ; 1131: 131-161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646509

RESUMO

Calcium (Ca2+) is a fundamental regulator of cell fate and intracellular Ca2+ homeostasis is crucial for proper function of the nerve cells. Given the complexity of neurons, a constellation of mechanisms finely tunes the intracellular Ca2+ signaling. We are focusing on the sarco/endoplasmic reticulum (SR/ER) calcium (Ca2+)-ATPase (SERCA) pump, an integral ER protein. SERCA's well established role is to preserve low cytosolic Ca2+ levels ([Ca2+]cyt), by pumping free Ca2+ ions into the ER lumen, utilizing ATP hydrolysis. The SERCA pumps are encoded by three distinct genes, SERCA1-3, resulting in 12 known protein isoforms, with tissue-dependent expression patterns. Despite the well-established structure and function of the SERCA pumps, their role in the central nervous system is not clear yet. Interestingly, SERCA-mediated Ca2+ dyshomeostasis has been associated with neuropathological conditions, such as bipolar disorder, schizophrenia, Parkinson's disease and Alzheimer's disease. We summarize here current evidence suggesting a role for SERCA in the neurobiology of neuropsychiatric and neurodegenerative disorders, thus highlighting the importance of this pump in brain physiology and pathophysiology.


Assuntos
Encéfalo , Retículo Endoplasmático , Doenças do Sistema Nervoso , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Encéfalo/enzimologia , Encéfalo/patologia , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
2.
Adv Exp Med Biol ; 1159: 49-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502199

RESUMO

The majority of enzymes in the sphingolipid (SL) biosynthetic pathway have been identified over the past couple of decades. Despite significant work, and despite their crucial and central roles in SL synthesis, significant information is still lacking concerning the enzymes that catalyze the N-acylation of sphingoid long chain bases, namely the ceramide synthases (CerS), a family of six mammalian genes originally named longevity assurance (Lass) genes. Each of these six endoplasmic reticulum (ER) membrane-bound enzymes utilizes a relatively restricted sub-set of fatty acyl-CoAs for N-acylation, but are far more promiscuous about the use of long chain bases. The reason that mammals and other species have multiple CerS, generating a specific subset of ceramides, is not yet known, but implies an important role for ceramides containing specific fatty acids in cell physiology. In this brief chapter, we will stroll down the CerS lane and discuss what is known, and what is not known, about this important enzyme family.


Assuntos
Ceramidas/biossíntese , Retículo Endoplasmático/enzimologia , Esfingosina N-Aciltransferase/fisiologia , Animais , Ácidos Graxos/química , Esfingolipídeos
3.
Genome Biol ; 20(1): 156, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387610

RESUMO

BACKGROUND: Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m6A in this process and the interplay between 5mC and m6A remain uncharacterized. RESULTS: Here, we show that mRNA m6A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m6A methylation is a prevalent modification in the mRNA of tomato fruit, and the m6A sites are enriched around the stop codons and within the 3' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m6A levels, while only 134 transcripts show decreased m6A enrichment, suggesting a global increase in m6A. The m6A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m6A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m6A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. CONCLUSIONS: Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m6A methylation during fruit ripening.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Metilação de DNA , Retículo Endoplasmático/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/metabolismo , Metilação , Mutação , Motivos de Nucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
4.
Mol Cell ; 74(1): 45-58.e7, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30846317

RESUMO

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Glicerídeos/biossíntese , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Lipogênese , Células 3T3 , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proliferação de Células , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Glicerol-3-Fosfato O-Aciltransferase/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Camundongos , Ácido Palmítico/toxicidade , Ligação Proteica
5.
Mol Biol Cell ; 30(9): 1069-1084, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30785834

RESUMO

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Homeostase , ATPases do Tipo-P/metabolismo , Filogenia , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
6.
Artigo em Inglês | MEDLINE | ID: mdl-30797055

RESUMO

Endoplasmic reticulum resident protein 44 (ERp44) is a protein disulfide isomerase (PDI) and a member of thioredoxin family, which is involved in several functions such as oxidative folding and polymerization of molecules, carrier protein activity, regulation of the Ca2+ ion levels in the endoplasmic reticulum (ER), cellular signaling, and maintenance of lumen redox homeostasis. In this study, ERp44 from Hippocampus abdominalis, commonly known as the big belly seahorse, was characterized. ERp44 possessed three PDI-like domains and one thioredoxin fold with a CXXC conserved motif. The open reading frame consisted of 1233 bp encoding 410 amino acids. Additionally, it contained a C-terminal RDEL motif, which suggests a localization of ERp44 to the endoplasmic reticulum. ShERp44 showed highest mRNA expression in the ovary, brain, and gills. Temporal expression of ShERp44 in blood showed significant upregulation against bacterial, LPS, and PolyI:C stimuli at 24 and 72 h. Trunk kidney tissue exhibited upregulated ShERp44 expression at 24 h in response to lipopolysaccharides and Streptococcus iniae and at 72 h in response to Edwardsiella tarda and poly I:C. NADPH turnover was observed as 0.06122 ±â€¯0.0075 µmol/s protein/µg through the HED assay. Insulin aggregation assay showed a significant reduction ability of rShERp44 by precipitating insulin rapidly, beginning at 5 min. Moreover, rShERp44-treated fathead minnow cells showed significant cell survival against 2-hydroxyethyl disulfide and thus exhibited capability to resist oxidative stress. Taken together, these findings provide insight into teleost defense mechanisms and functional properties of ERp44 in controlling redox homeostasis at the molecular level.


Assuntos
Retículo Endoplasmático , Proteínas de Peixes , Estresse Oxidativo/fisiologia , Isomerases de Dissulfetos de Proteínas , Smegmamorpha , Motivos de Aminoácidos , Animais , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Regulação da Expressão Gênica/fisiologia , Rim/enzimologia , Especificidade de Órgãos/fisiologia , Oxirredução , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética , Domínios Proteicos , Smegmamorpha/genética , Smegmamorpha/metabolismo
7.
Circ Res ; 124(1): 79-93, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30582446

RESUMO

RATIONALE: Endoplasmic reticulum (ER) stress dysregulates ER proteostasis, which activates the transcription factor, ATF6 (activating transcription factor 6α), an inducer of genes that enhance protein folding and restore ER proteostasis. Because of increased protein synthesis, it is possible that protein folding and ER proteostasis are challenged during cardiac myocyte growth. However, it is not known whether ATF6 is activated, and if so, what its function is during hypertrophic growth of cardiac myocytes. OBJECTIVE: To examine the activity and function of ATF6 during cardiac hypertrophy. METHODS AND RESULTS: We found that ER stress and ATF6 were activated and ATF6 target genes were induced in mice subjected to an acute model of transverse aortic constriction, or to free-wheel exercise, both of which promote adaptive cardiac myocyte hypertrophy with preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO [conditional knockout]) blunted transverse aortic constriction and exercise-induced cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for ATF6 in compensatory myocyte growth. Transcript profiling and chromatin immunoprecipitation identified RHEB (Ras homologue enriched in brain) as an ATF6 target gene in the heart. RHEB is an activator of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a major inducer of protein synthesis and subsequent cell growth. Both transverse aortic constriction and exercise upregulated RHEB, activated mTORC1, and induced cardiac hypertrophy in wild type mouse hearts but not in ATF6 cKO hearts. Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked phenylephrine- and IGF1 (insulin-like growth factor 1)-mediated RHEB induction, mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB expression. Moreover, adeno-associated virus 9- RHEB restored cardiac growth to ATF6 cKO mice subjected to transverse aortic constriction. Finally, ATF6 induced RHEB in response to growth factors, but not in response to other activators of ATF6 that do not induce growth, indicating that ATF6 target gene induction is stress specific. CONCLUSIONS: Compensatory cardiac hypertrophy activates ER stress and ATF6, which induces RHEB and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth stimuli and mTORC1-mediated cardiac growth.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Hipertrofia Ventricular Esquerda/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Miócitos Cardíacos/enzimologia , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Ativação Transcricional , Função Ventricular Esquerda , Remodelação Ventricular , Fator 6 Ativador da Transcrição/deficiência , Fator 6 Ativador da Transcrição/genética , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Retículo Endoplasmático/enzimologia , Estresse do Retículo Endoplasmático , Predisposição Genética para Doença , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/patologia , Fenótipo , Dobramento de Proteína , Proteostase , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Transdução de Sinais
8.
Toxicol Mech Methods ; 28(7): 539-546, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29708463

RESUMO

Quantum dots (QDs) have widespread application in many fields such as medicine and electronics. The need for understanding the potentially harmful side effects of these materials becomes clear. In this study, the toxicity of cadmium telluride quantum dots (CdTe-QDs) and bulk Cd2+ has been investigated and compared by applying metabolomics methods. The datasets were 1H-NMR data from mice plasma which had been taken from four groups of mice in different time intervals. Then, the data were analyzed by applying chemometrics methods and the metabolites were found from Human Metabolome Database (HMDB). The results showed the significant change in the level of some metabolites especially estrogenic steroids in different groups with different amounts of received Cd. The findings also indicated that steroid hormone biosynthesis, lysine biosynthesis and taurine and hypotaurine metabolism are the most affected pathways by CdTe-QDs especially in estrogenic steroids. The over-representation analysis indicated that endoplasmic reticulum, gonads, and hepatocytes are most affected. Since the pattern of metabolite alteration of CdTe-QDs with equivalent Cd2+ was similar to those of CdCl2, it was postulated that beside Cd2+ effects, the toxicity of CdTe-QDs is associated with other factors.


Assuntos
Compostos de Cádmio/toxicidade , Retículo Endoplasmático/efeitos dos fármacos , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pontos Quânticos/toxicidade , Telúrio/toxicidade , Testículo/efeitos dos fármacos , Animais , Cádmio/química , Cádmio/toxicidade , Compostos de Cádmio/administração & dosagem , Compostos de Cádmio/química , Intoxicação por Cádmio/enzimologia , Intoxicação por Cádmio/metabolismo , Intoxicação por Cádmio/patologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Injeções Intraperitoneais , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Metabolômica/métodos , Camundongos , Especificidade de Órgãos , Tamanho da Partícula , Análise de Componente Principal , Pontos Quânticos/administração & dosagem , Distribuição Aleatória , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Propriedades de Superfície , Telúrio/administração & dosagem , Telúrio/química , Testículo/metabolismo , Testículo/patologia , Testes de Toxicidade Crônica
9.
PLoS Pathog ; 14(4): e1007029, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29709018

RESUMO

Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.


Assuntos
Retículo Endoplasmático/virologia , Rim/enzimologia , Lipídeos de Membrana/metabolismo , Fosfolipases A2/metabolismo , Replicação Viral , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Células Cultivadas , Cercopithecus aethiops , Cricetinae , Retículo Endoplasmático/enzimologia , Rim/virologia , Células Vero , Febre do Nilo Ocidental/enzimologia
10.
Adv Exp Med Biol ; 1062: 265-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29845539

RESUMO

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and ß-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.


Assuntos
Retículo Endoplasmático/enzimologia , Viroses/enzimologia , Viroses/imunologia , Fenômenos Fisiológicos Virais , alfa-Glucosidases/química , alfa-Glucosidases/imunologia , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/virologia , Interações Hospedeiro-Patógeno , Humanos , Viroses/genética , Viroses/virologia , Vírus/genética , alfa-Glucosidases/genética
11.
Adv Exp Med Biol ; 1062: 277-301, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29845540

RESUMO

The antiviral mechanism of action of iminosugars against many enveloped viruses, including dengue virus (DENV), HIV, influenza and hepatitis C virus, is believed to be mediated by inducing misfolding of viral N-linked glycoproteins through inhibition of host endoplasmic reticulum-resident α-glucosidase enzymes. This leads to reduced secretion and/or infectivity of virions and hence lower viral titres, both in vitro and in vivo. Free oligosaccharide analysis from iminosugar-treated cells shows that antiviral activity correlates with production of mono- and tri-glucosylated sugars, indicative of inhibition of ER α-glucosidases. We demonstrate that glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. Galactose-mimicking iminosugars that have been tested do not inhibit glycoprotein processing but do inhibit glycolipid processing, and are not antiviral against DENV. By comparison, the antiviral activity of glucose-mimetic iminosugars that inhibit endoplasmic reticulum-resident α-glucosidases, but not glycolipid processing, demonstrates that inhibition of α-glucosidases is responsible for iminosugar antiviral activity against DENV. This monograph will review the investigations of many researchers into the mechanisms of action of iminosugars and the contribution of our current understanding of these mechanisms for optimising clinical delivery of iminosugars. The effects of iminosugars on enzymes other than glucosidases, the induction of ER stress and viral receptors will be also put into context. Data suggest that inhibition of α-glucosidases results in inhibited release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.


Assuntos
Vírus da Dengue/fisiologia , Dengue/imunologia , Açúcares/imunologia , Animais , Dengue/enzimologia , Dengue/genética , Dengue/virologia , Vírus da Dengue/genética , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/virologia , Humanos , alfa-Glucosidases/genética , alfa-Glucosidases/imunologia
12.
Food Chem ; 256: 31-39, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29606453

RESUMO

This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry.


Assuntos
Pão/análise , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Sequência de Bases , China , Dicroísmo Circular , Retículo Endoplasmático/enzimologia , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Peso Molecular , Oxirredutases/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Espectrofotometria
13.
Diabetes ; 67(6): 1128-1139, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29563152

RESUMO

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of ß-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca2+]i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca2+]i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca2+]i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that ß-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca2+]i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca2+ release from the acidic stores and further amplified by Ca2+-induced Ca2+ release from the sarco/endoplasmic reticulum.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Epinefrina/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Regulação para Cima , Neurônios Adrenérgicos/citologia , Neurônios Adrenérgicos/efeitos dos fármacos , Neurônios Adrenérgicos/metabolismo , Animais , Animais não Endogâmicos , Canais de Cálcio/química , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/efeitos dos fármacos , Pâncreas/inervação , Pâncreas/metabolismo , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/metabolismo , Técnicas de Cultura de Tecidos , Regulação para Cima/efeitos dos fármacos
14.
BMC Plant Biol ; 18(1): 41, 2018 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-29506473

RESUMO

BACKGROUND: The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between the plant and the environment. The cuticle is composed of cutin and wax. Cuticular wax plays an important role in the survival of plants by serving as the interface between plants and their biotic and abiotic environments, especially restricting nonstomatal water loss. Leaf cuticular waxes of hexaploid wheat at the seedling stage mainly consist of primary alcohols, aldehydes, fatty acids, alkane and esters. Primary alcohols account for more than 80% of the total wax load. Therefore, we cloned several genes encoding fatty acyl-coenzyme A reductases from wheat and analyzed their function in yeast and plants. We propose the potential use of these genes in wheat genetic breeding. RESULTS: We reported the cloning and characterization of three TaFARs, namely TaFAR6, TaFAR7 and TaFAR8, encoding fatty acyl-coenzyme A reductases (FAR) in wheat leaf cuticle. Expression analysis revealed that TaFAR6, TaFAR7 and TaFAR8 were expressed at the higher levels in the seedling leaf blades, and were expressed moderately or weakly in stamen, glumes, peduncle, flag leaf blade, sheath, spike, and pistil. The heterologous expression of three TaFARs in yeast (Saccharomyces cerevisiae) led to the production of C24:0 and C26:0 primary alcohols. Transgenic expression of the three TaFARs in tomato (Solanum lycopersicum) and rice (Oryza sativa) led to increased accumulation of C24:0-C30:0 primary alcohols. Transient expression of GFP protein-tagged TaFARs revealed that the three TaFAR proteins were localized to the endoplasmic reticulum (ER), the site of wax biosynthesis. The three TaFAR genes were transcriptionally induced by drought, cold, heat, powdery mildew (Blumeria graminis) infection, abscisic acid (ABA) and methyl jasmonate (MeJa) treatments. CONCLUSIONS: These results indicated that wheat TaFAR6, TaFAR7 and TaFAR8 are involved in biosynthesis of very-long-chain primary alcohols in hexaploid wheat and in response to multiple environmental stresses.


Assuntos
Aldeído Oxirredutases/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Plantas/metabolismo , Poliploidia , Triticum/enzimologia , Ceras/metabolismo , Ácido Abscísico/farmacologia , Acetatos/farmacologia , Aldeído Oxirredutases/genética , Ciclopentanos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Oxilipinas/farmacologia , Proteínas de Plantas/genética , Triticum/efeitos dos fármacos , Triticum/genética
15.
Parasitol Res ; 117(5): 1599-1611, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29594345

RESUMO

Endoplasmic reticulum (ER)-associated degradation (ERAD) and unfolded protein response (UPR) pathways are important for quality and quantity control of membrane and secretory proteins. We have identified orthologs of ER-associated ubiquitin conjugating enzymes (E2s) Ubc6/Ube2j2 and Ubc7/Ube2g2, ubiquitin ligases (E3) Hrd1 and GP78/AMFR, and sensor of UPR, Ire1 in E. histolytica that show conservation of important features of these proteins. Biochemical characterization of the ortholog of ERAD E2, Ubc7/Ube2g2 (termed as EhUbc7), was carried out. This E2 was transcriptionally upregulated several folds upon induction of UPR with tunicamycin. Ire1 ortholog was also upregulated upon UPR induction suggesting a linked UPR and ERAD pathway in this organism. EhUbc7 showed enzymatic activity and, similar to its orthologs in higher eukaryotes, formed polyubiquitin chains in vitro and localized to both cytoplasm and membranes. However, unlike its ortholog in higher eukaryotes, it also showed localization to the plasma membrane along with calreticulin. Inactivation of EhUbc7 significantly inhibited erythrophagocytosis, suggesting a novel function that has not been reported before for this E2. No change in growth, motility, or cell-surface expression of Gal/GalNAC lectin was observed due to inactivation of EhUbc7. The protein was present in the phagocytic cups but not in the phagosomes. A significant decrease in the number of phagocytic cups in inactive EhUbc7 expressing cells was observed, suggesting altered kinetics of phagocytosis. These findings have implications for evolutionary and mechanistic understanding of connection between phagocytosis and ER-associated proteins.


Assuntos
Membrana Celular/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/enzimologia , Entamoeba histolytica/enzimologia , Fagocitose/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Calreticulina/metabolismo , Endorribonucleases , Entamoeba histolytica/metabolismo , Humanos , Proteínas Serina-Treonina Quinases , Receptores do Fator Autócrino de Motilidade , Tunicamicina/farmacologia , Ubiquitina/metabolismo
16.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(6): 614-624, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29526665

RESUMO

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells. RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66 µm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally. In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.


Assuntos
Coenzima A Ligases/metabolismo , Retículo Endoplasmático/enzimologia , Gotículas Lipídicas/enzimologia , Triglicerídeos/biossíntese , Linhagem Celular Tumoral , Humanos
17.
Mol Cell Biochem ; 448(1-2): 275-286, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29450800

RESUMO

Fatty acid uptake and metabolism are often dysregulated in cancer cells. Fatty acid activation is a critical step that allows these biomolecules to enter cellular metabolic pathways such as mitochondrial ß-oxidation for ATP generation or the lipogenic routes that generate bioactive lipids such as the inositol phospholipids. Fatty acid activation by the addition of coenzyme A is catalysed by a family of enzymes called the acyl CoA synthetase ligases (ACSL). Furthermore, enhanced expression of particular ACSL isoforms, such as ACSL4, is a feature of some more aggressive cancers and may contribute to the oncogenic phenotype. This study focuses on ACSL3 and ACSL4, closely related structural homologues that preferentially activate palmitate and arachidonate fatty acids, respectively. In this study, immunohistochemical screening of multiple soft tissue tumour arrays revealed that ACSL3 and ACSL4 were highly, but differentially, expressed in a subset of leiomyosarcomas, fibrosarcomas and rhabdomyosarcomas, with consistent cytoplasmic and granular stainings of tumour cells. The intracellular localisations of endogenously expressed ACSL3 and ACSL4 were further investigated by detailed subcellular fractionation analyses of HT1080 fibrosarcoma and MCF-7 breast cancer cells. ACSL3 distribution closely overlapped with proteins involved in trafficking from the trans-Golgi network and endosomes. In contrast, the ACSL4 localisation pattern more closely followed that of calnexin which is an  endoplasmic reticulum resident chaperone. Confocal immunofluorescence imaging of MCF-7 cells confirmed the intracellular localisations of both enzymes. These observations reveal new information regarding the compartmentation of fatty acid metabolism in cancer cells.


Assuntos
Neoplasias da Mama/enzimologia , Coenzima A Ligases/metabolismo , Retículo Endoplasmático/enzimologia , Endossomos/enzimologia , Fibrossarcoma/enzimologia , Proteínas de Neoplasias/metabolismo , Rede trans-Golgi/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Coenzima A Ligases/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Endossomos/genética , Endossomos/patologia , Feminino , Fibrossarcoma/genética , Fibrossarcoma/patologia , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Rede trans-Golgi/genética , Rede trans-Golgi/patologia
18.
Subcell Biochem ; 87: 229-258, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29464562

RESUMO

The calcium pump (a.k.a. Ca2+-ATPase or SERCA) is a membrane transport protein ubiquitously found in the endoplasmic reticulum (ER) of all eukaryotic cells. As a calcium transporter, SERCA maintains the low cytosolic calcium level that enables a vast array of signaling pathways and physiological processes (e.g. synaptic transmission, muscle contraction, fertilization). In muscle cells, SERCA promotes relaxation by pumping calcium ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR), the main storage compartment for intracellular calcium. X-ray crystallographic studies have provided an extensive understanding of the intermediate states that SERCA populates as it progresses through the calcium transport cycle. Historically, SERCA is also known to be regulated by small transmembrane peptides, phospholamban (PLN) and sarcolipin (SLN). PLN is expressed in cardiac muscle, whereas SLN predominates in skeletal and atrial muscle. These two regulatory subunits play critical roles in cardiac contractility. While our understanding of these regulatory mechanisms are still developing, SERCA and PLN are one of the best understood examples of peptide-transporter regulatory interactions. Nonetheless, SERCA appeared to have only two regulatory subunits, while the related sodium pump (a.k.a. Na+, K+-ATPase) has at least nine small transmembrane peptides that provide tissue specific regulation. The last few years have seen a renaissance in our understanding of SERCA regulatory subunits. First, structures of the SERCA-SLN and SERCA-PLN complexes revealed molecular details of their interactions. Second, an array of micropeptides concealed within long non-coding RNAs have been identified as new SERCA regulators. This chapter will describe our current understanding of SERCA structure, function, and regulation.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio , Retículo Endoplasmático , Células Musculares/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Cálcio/química , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cristalografia por Raios X , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteolipídeos/química , Proteolipídeos/genética , Proteolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
19.
Dev Cell ; 44(2): 261-270.e6, 2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29290583

RESUMO

Spatial organization of phospholipid synthesis in eukaryotes is critical for cellular homeostasis. The synthesis of phosphatidylcholine (PC), the most abundant cellular phospholipid, occurs redundantly via the ER-localized Kennedy pathway and a pathway that traverses the ER and mitochondria via membrane contact sites. The basis of the ER-mitochondrial PC synthesis pathway is the exclusive mitochondrial localization of a key pathway enzyme, phosphatidylserine decarboxylase Psd1, which generates phosphatidylethanolamine (PE). We find that Psd1 is localized to both mitochondria and the ER. Our data indicate that Psd1-dependent PE made at mitochondria and the ER has separable cellular functions. In addition, the relative organellar localization of Psd1 is dynamically modulated based on metabolic needs. These data reveal a critical role for ER-localized Psd1 in cellular phospholipid homeostasis, question the significance of an ER-mitochondrial PC synthesis pathway to cellular phospholipid homeostasis, and establish the importance of fine spatial regulation of lipid biosynthesis for cellular functions.


Assuntos
Carboxiliases/metabolismo , Retículo Endoplasmático/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Fosfatidiletanolaminas/metabolismo , Carboxiliases/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Homeostase , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Sinais Direcionadores de Proteínas
20.
Toxicol In Vitro ; 47: 26-37, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29107685

RESUMO

A library of N-protected dehydroamino acids, namely dehydroalanine, dehydroaminobutyric acid and dehydrophenylalanine derivatives, was screened in three human cancer cell lines [(lung (A549), gastric (AGS) and neuroblastoma (SH-SY5Y)] in order to characterize their toxicological profile and identify new molecules with potential anticancer activity. Results showed N-protected dehydrophenylalanine and dehydroaminobutyric acid derivatives have no or low toxicity for all tested cell lines. The N-protected dehydroalanines exhibit significant toxic effects and the AGS and SH-SY5Y cells were significantly more vulnerable than A549 cells. Four α,ß-dehydroalanine derivatives, with IC50<62.5µM, were selected to investigate the pathways by which these compounds promote cell death. All compounds, at their IC50 concentrations, were able to induce apoptosis in both AGS and SH-SY5Y cell lines. In both cell lines, loss of mitochondrial membrane potential (ΔΨm) was found and caspase activity was increased, namely endoplasmic reticulum-resident caspase-4 in AGS cells and caspase-3/7 in SH-SY5Y cells. When evaluated in a non-cancer cell line, the molecules displayed no to low toxicity, thus suggesting some degree of selectivity for cancer cells. The results indicate that α,ß-dehydroalanine derivatives can be considered a future resource of compounds able to work as anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Ácido gama-Aminobutírico/análogos & derivados , Alanina/efeitos adversos , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Caspases/química , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Descoberta de Drogas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fenilalanina/efeitos adversos , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Bibliotecas de Moléculas Pequenas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Relação Estrutura-Atividade , Ácido gama-Aminobutírico/efeitos adversos , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/farmacologia
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